University of Montana ScholarWorks at University of Montana
Graduate Student Theses, Dissertations, & Professional Papers Graduate School
1981
Some antifungal activities of guinea pig peritoneal exudate cells against Sporothrix schenckii
Daryl S. Paulson The University of Montana
Follow this and additional works at: https://scholarworks.umt.edu/etd Let us know how access to this document benefits ou.y
Recommended Citation Paulson, Daryl S., "Some antifungal activities of guinea pig peritoneal exudate cells against Sporothrix schenckii" (1981). Graduate Student Theses, Dissertations, & Professional Papers. 7325. https://scholarworks.umt.edu/etd/7325
This Thesis is brought to you for free and open access by the Graduate School at ScholarWorks at University of Montana. It has been accepted for inclusion in Graduate Student Theses, Dissertations, & Professional Papers by an authorized administrator of ScholarWorks at University of Montana. For more information, please contact [email protected]. COPYRIGHT ACT OF 1976
This is an unpublished manuscript in which copyright sub s i s t s . Any further reprinting of its contents must be approved BY THE AUTHOR.
Mansfield Library University of Montana DATE:
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. SOME ANTIFUNGAL ACTIVITIES OF
GUINEA PIG PERITONEAL EXUDATE CELLS
AGAINST
SPOROTHRIX SCHENCKII
by
Daryl S Paulson
B.A., University of Montana, 1972
Presented in partial fulfillment of the requirements for the degree of
Master of Science
UNIVERSITY OF MONTANA
1981
Approved by:
airman ers
Dean, Graduate Schoo
Date
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: EP38126
All rights reserved
INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted.
In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. UMI DisM rtfltion PuUiahing
UMI EP38126 Published by ProQuest LLC (2013). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code
ProQuest
ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 106 -13 46
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Paulson, Daryl S., M.S., 1977 Microbiology
Some ancifungal activities of guinea pig peritoneal exudate cells against Sporothrix schenckii. (46 pp.)
Director; John J. Taylor
Six guinea pig models were treated with schenckii conidia in various combinations to ascertain the effects of peritoneal exudate cells (PECs) on these conidia, to find whether or not those PEC effects were fungicidal or fungistatic to _S. schenckii conidia, and to determine the effects hydrocortisone acetate compromise had upon PEC activity. Phagocytic res ponses were measured using these 6 animal models which included animals immunized with viable conidia, animals immunized with non-viable conidia, untreated controls, animals compromised with cortisone but not immunized, animals both compromised and immunized with viable conidia and finally animals both compromised and immunized with non-viable conidia. Statisti cal analyses were used to compare the groups. It appears that animals immunized with viable conidia are more efficient in clearing these fungi via phagocytosis, that phagocytic rate and fungicidal properties are posi tively correlated, and that cortisone compromise, achieved through repeated cortisone injections into these animals, reduces both phagocytic ability and fungicidal properties to PECs.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENTS
This work is dedicated to my two friends and fellow Marines, John
Burke and John Osterhaus, who were killed in action at An Hoa Fire Base,
South Vietnam, on Operation Taylor Common during the Tet offensive, 1968.
Special thanks to Dr. John J. Taylor for his guidance in this work and
to the other members of my committee, Drs. Mitsuru J. Nakamura, Clarence
A, Speer, and Todd G. Cochran. I also extend my appreciation to Dr. Jon
A. Rudbach for his valuable assistance in opening the initial computer
account for the Microbiology Department, which allowed me to greatly
sophisticate the statistical computations required in the multivariate
analyses of this work. Also my thanks to Drs. Rudy Gideon and Don O.
Loftsgaarden of the Mathematics Department for their statistical con
sultations .
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS
Page ABSTRACT ...... ü
ACKNOWLEDGEMENT...... H i
TABLE OF CONTENTS ...... iv
LIST OF TABLES ...... v
LIST OF FIGURES ...... vi
Chapter
I. INTRODUCTION ...... 1
II. MATERIALS AND METHODS ...... 14
Animal Models ...... 14
Fungal cultures ...... 14
Tissue culturing media ...... 15
G l a s s w a r e ...... 16
Antigen preparations ...... 16
Experimental m o d e l s ...... 17
In vitro methodology...... 18
Quantitative analysis ...... 21
III. R E S U L T S ...... 22
IV. DISCUSSION AND CONCLUSIONS...... 34
V. S U M M A R Y ...... 42
REFERENCES CITED ...... 4 3
IV
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES
Table Page
1. Average number of conidia phagocytosed per PEC based on 20 randomly selected P E C s ...... 25
2. Average number of conidia phagocytosed per PEC based on only those PECs containing conidia...... 26
3. ANOVA summary table of the average number of conidia phagocytosed per 20 randomly selected PECs ...... 27
4. Summary table of the nested ANOVA design based on phagocytic efficiency on t i m e ...... 28
5. ANOVA summary table for average numbers of conidia phagocytosed for each model regressed on (X^) treatment, and contrasted with the addition of (X-) age, and/or (Xg) sex...... 29
V Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES
Figure Page
1. Average number of conidia phagocytosed per PEC based on 20 randomly selected P E C s ...... 23
2. Average number of conidia phagocytosed per PEC based on only those PECs containing conidia ...... 24
vx
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER I
INTRODUCTION
A. BASIC REVIEW MATERIAL
Host/Parasite Relationship
For man and other animals, parasitic microbial Infection Is the
Invasion of the host’s tissue by a multiplying microorganism which
consumes the host’s nutrients, placing a strain on the host’s homeostatic
ability. However, through the course of evolution, several effective
and efficient antimicrobial defense systems have evolved: the Immune
system and the reticuloendothelial system which are Interfunctional.
Immune Sys t em
The immune system(47) composed of primary and secondary lymphoid
tissue (spleen, thymus, lymph nodes, bone marrow, lymphocytes and Peyer’s
patches) can be further divided into two cell type systems: the humoral
and the cellular Immune systems. The humoral system refers to those
lymphoid cells whose primary function is to produce antibodies (glyco
proteins) capable of binding non-covalently through electrostatic forces,
van der Walls force, hydrogen bonding of hydrophobic/hydrophilic Inter
actions with specific complementary antigens. These humoral cells (B cells)
are bone marrow derived and are the precursor cells of Immunoglobulin
secreting cells or plasma cells. The cellular lymphoid system consists of
thymus derived lymphoid cells (T cells), which do not differentiate Into
immunoglobulin secreting cells, but Instead produce lymphokines such as
macrophage activation factors, macrophage inhibition factors, lympho-
cytotoxlns. Interferon, activation factors for eliciting the humoral
Immune response and chemotactic factors. They are responsible for hetero-
- 1- Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. — 2 —
genous tissue graft rejection, they have the ability to directly lyse
and destroy foreign cells Including certain tumors, and they are Involved
In delayed hypersensitivity reactions.
Reticuloendothelial System
The reticuloendothelial system(5,44) Is composed of a network of highly
phagocytic reticular cells lining organ tissues such as the spleen, the
thymus, the liver (Kupffer’s cells) and the vascular endothelium of the
tunica interna. In addition to these sessile cells, motile phagocytic
cells occur In the peripheral pools as neutrophils, basophils, eosinophils,
and monocytes. The macrophage, the end cell of the monocyte line and the
phagocyte found In various tissue, Is free to wander by ameboid movement
through tissue. It is especially concentrated within the loose connective
tissue, but Is not restricted to that tissue. Specialized macrophages are
found In the alveoli of the lungs (alveolar macrophages) and In the
peritoneal cavity as peritoneal macrophages.
Peritoneal Exudate Cells
The cells of special Interest In this thesis are the peritoneal exu
date cells (PEC) which Include PMNs, monocyte/macrophages, and lymphocytes
but particularly the macrophage since It Is the most abundant cell normally
found In the extravascular area of the peritoneal cavity.
Macrophage: The macrophage, derived from the circulating white blood
cell, the monocyte. Is a long lived and a very effective phagocytic cell
In mammals(13,39,42). Electron microscopy has revealed that a unit mem
brane exists In the macrophage which has many protuberances (microvilli)
and Invaginations. The cytoplasm contains smooth and rough endoplasmic
reticulum. IVo types of vacuoles have been shown to exist: a smaller
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. pinocytic vesicle and a larger phagocytic vacuole. The macrophage ingests
particles smaller than 0 .1pm by pinocytosis and engulfs particles of
larger size through phagocytosis. It has a plasma membrane which is capable
of recongnizing two of four subclasses of the human gamma immunoglobulin
class, IgG^ and IgGg which differ only in their physicochemical composition
in the constant globulin region(4). It can also recognize the complement
fraction C3b. The macrophage has been shown to be capable of both non-
immunologically and immunologically controlled phagocytesis/pinocytosis.
The macrophage has also been shown to attach to the Fc portion of
the gamma immunoglobulin, which has previously been bound to an antigen.
During phagocytosis, a particle is bound to the macrophage’s membranee
receptors and surrounded by the cell membrane. The particle is then
internalized into a phagosome and finally digested after the phagosome
containing the particle and a lysosome have been fused(45).
Metabolism of the macrophage is dependent upon the activation state
of the cell. An unactivated macrophage obtains energy primarily through
anaerobic glycolysis but the activated macrophage has a significant increase
in the hexose monophosphate shunt- A macrophage may become activated by
exposure to lymphokines produced by sensitized T cells or by direct antigen
contact. An activated macrophage is larger than the unactivated form, an
activated macrophage adheres to glass more readily than an unactivated
macrophage, and an activated macrophage is more phagocytic than an unact
ivated type especially in phagocytosing complement-coated C3b, or IgG^ and
IgGj coated particles. An activated macrophage also exhibits a greater
chemotactic ability than the non-activated type.
Mechanisms of intracellular killing are not as well known in macrophages
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. -4-
as in neutrophilic degradation involving myeloperoxidase» an acid pH, a
halogen, and hydrogen peroxide. However, lysozyme and hydrogen peroxide
are known to be of importance in the killing and degradationability by
the macrophage.
The killing effect of the macrophage is related to the engulfed
particle's chemical structure and to the activation of the macrophage.
The activated macrophage is especially efficacious in phagocytosing intra
cellular organisms.
Neutrophil; The neutrophil, a phagocytic leukocyte(8 ), is also
important as a PEC. The neutrophil, a polymorphonuclear leukocyte,
accounts for about 60% of the total number of white blood cells contained
in the peripheral blood pool, and is of secondary importance to the macro
phage in this thesis only because it is not the major phagocytic cell
line found in the peritoneal cavity. The neutrophil is a key cell in the
phagocytic defense system because of its phagocytic effectiveness and
efficiency and its quick response to microbial pathogens.
The neutrophil is 10-2Qium in diameter and is distinguished by its
3-to-5 lobed nucleus held together by the nuclear envelope. Its average
life span is 4 to 5 days and therefore, it is a short-lived cell. The
neutrophil is extremely motile via ameboid movement and may become
.chemotactic in the presence of certain attracting microbial products,
tissue proteases, and C3b and C4b complement components. Opsonized micro
bial organisms are more readily phagocytosed than non-opsonized ones and
neutrophils having surface Fc receptors which recognize opsonins are very
effective in phagocytosing opsonized microbial organisms in one of three
ways(5,12): (1) by a specific IgG antibody/antigen complex; (2) by a
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. -5—
specific IgG antibody/complement complex; or (3) by a non-specific compiiement
fraction activated by a bacterial or fungal polysaccharide which forms iaii
microbe/complement complex evoking phagocytosis. >ln
Phagocytosis of microbial organisms Is also educed by three additional
factors: (1 ) by the proximity of an antigen and a neutrophil relative tmC
each other since the more packed together they are, the greater the pha*d
gocytlc activity Independent of chemotactic or opsonizing factors; (2)>r t
through natural antibodies other than IgG molecules synthesized for spœn?-
Iflc antigens ; and (3) through leukoklnln, a type of IgG complex which Ic
can coat the neutrophil’s surface stimulating neutrophilic phagocytoslsicu
Ostensibly, the biological activity of leukoklnln Is dependent upon a Iv'j
single peptide chain (Thr-Lys-Pro-Arg) named Tuftsin since It was dlsco%sr-
ed at Tufts University(25).
The energy requisite for the neutrophil’s killing mechanism Is proch,'
vlded by the glycolytic pathway. This pathway Is significant because
neutrophilic phagocytosis often occurs In hypoxic surroundings such as n o
abscessed lesions.
Once a microbe and a neutrophil have made contact, pseudopodia rop
from the neutrophil extend and fuse on the far side of the microbe, encs&T-
Ing It within a phagosome. The phagosome containing the microbe moves a p
toward the cell’s center where It fuses with lysosomes (at a low pH)■coiâi
talnlng myeloperoxidase, hydrogen peroxide, and a halide. Once fusion gif'
these constituents has been completed, the microbe Is killed and degradiqd!.
This Is an especially effective mechanism In phagocytic removal of e ,
Intercellular pathogens.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. —6—
B. SPECIFIC REVIEW MATERIAL
Three Host/Parasite Models
Bacterial Model: The polymorphonuclear leukocyte is usually the first
cell to provide defense against pathogenic bacteria(4). Upon entry into
a host, specific bacterial end products are chemotactic for neutrophils.
Once a neutrophil has come in contact with a bacterial cell, the bacterium
is phagocytosed. The phagocytic efficiency is enhanced if the bacterial
cell has been previously opsonized, for example, by IgG antibody.
If the bacterial pathogen is a long-lived and/or an intracellular
organism, the activated macrophage is more effective in its removal than
the neutrophil. Mackaness(34) studied extensively the macrophage's role
in intracullular bacterial infection. He introduced, intravenously, a
sub-lethal dose of virulent Listeria monocytogenes into a mouse model
and ascertained that it was initially removed by neutrophils and sessile
cells of the reticuloendothelial system. However, monocytogenes was
found to continue multiplying in these cells for 3 to 4 days after being
phagocytosed. But after the 4th day, the bacterial population dropped
off rapidly until monocytogenes was eliminated from the infected mice.
The mice proved to be highly resistant to a rechallenge of these bacteria
and suffered no pathological effects with a rechallenge of up to a LD^q
dose of monocytogenes.
The reason for the high level of resistance was that the bacterial
antigens stimulated the cellular immune system, elicited by the T lympho
cytes, between the 1st and 2nd days of the challenge. The sensitized T
cells released lymphokines such as macrophage activation factors and
macrophage inhibition factors into the area where the T lymphocytes and
bacterial cells interacted. The lymphokines activated the macrophages
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. -7-
and they readily phagocytosed the bacteria. Resistance to monocytogenes
was long-lived in these mice due to the T memory cells which were capable
of reactivating the macrophages upon a rechallenge and subsequent recogni
tion of monocytogenes.
A different bacterial model, Mycobacterium tuberculosis, a long-lived,
acid fast bacterium and the causative agent of tuberculosis, has provided
similar results. With low level doses of M. tuberculosis in healthy, well
nourished mammals, the disease is usually averted through neutrophilic
phagocytosis. However, when M. tuberculosis is not completely destroyed
by neutrophils, the activated macrophages become a preeminent factor in
the phagocytosis of these bacteria.
In 1965, Lurie and Dannenburg(32), using inbred rabbits as hosts,
studied macronhage/M. tuberculosis interaction. They concluded that resis
tance or susceptibility to tuberculosis in the rabbits was dependent on
the macrophage’s phagocytosing ability. In addition, when they injected
cortisone into these rabbits, they found that the cortisone (which they
thought prevented the fusion of the macrophage’s phagosome and lysosome
bodies) severely reduced the macrophage’s ability to eliminate ^ tuberculosis,
However, Hart and Armstrong(18) reported in 1974 that tuberculosis survived
degradation in their study, even after being phagocytosed and both the
phagosomal and lysosomal bodies had in fact fused.
Moreover, it had previously been shown in several studies that the
cellular immune system accounted for a major portion of the phagocytic
elimination of M. tuberculosis. For example, in 1960, Hsu and Kapral(25)
compared guinea pig immune peritoneal macrophages with non-immune periton
eal macrophages. They concluded that the immune and therefore activated
macrophages were more effective in phagocytosing tuberculosis than
non-immune macrophages
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. — 8—
Youmans and Youmans in 1969(48) reported that M. tuberculosis was
not only eliminated faster by immunized (activated) macrophages than by
non-activated macrophages, but the activated macrophages, immunized
specifically for ^ tuberculosis were as expeditious in removing non
related organisms such as Lj_ monocytogenes as those macrophages sensitized
with monocytogenes.
In a bacterial model using Salmonella typhimurium, a gram negative
bacterium the causative agent of salmonella gastroenteritis, several
studies (3,35) concluded that macrophages activated by T lymphocytes were
more efficient in killing S. typhimurium than non-activated macrophages.
Also, it was reported that humoral components (B cells) were not a sign
ificant factor in the activation of the macrophages against _S_j_ typhimurium.
Viral Model; Since viral infections are intracellular, macrophages
may have an antiviral function in this type of infection (2,37). T
lymphocytes, upon recognition of viral antigen, release lymphokines which
in turn activate macrophages. These activated macrophages then pinocytose
the free virus particles. It has been demonstrated in vitro that activated
macrophages do restrict intracellular replication of some viruses through
phagocytoses. It has also been shown that activated macrophages protect
new born mice from herpesvirus infection(19). There is evidence that
macrophage-produced interferon offers resistance to general viral infections;
however, no conclusive evidence has been shown to date.
Fungal Model: Studies pertaining to subcutaneous and systemic fungal
infections in mammalian hosts have been neglected, for the most part, rela
tive to the extensive and intensive studies conducted upon bacterial and
viral models. One reason for this is the low invasiveness many fungi have
upon healthy and well nourished mammals. However, with the advent of
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. -9-
broad-spectrum antibiotics, synthetic anti-inflammatory drugs such as the
corticosteroids, and present oncological treatment methods (eg. chemo
therapy), the occurrence of fungal diseases such as candidiasis, mucoirmy-
cosis, cryptococcosis, and aspergillosis has been increasing significantly
(29.45).
Natural resistance appears to be a major factor in protecting the
host against pathogenic subcutaneous and systemic fungal infections.
Neutrophils rapidly provide initial protection against these organisms
until the macrophages become activated. Once activated, macrophages can
be effective antifungal agents especially in, for example, fungal infect
ions like Cryptococcus neoformans.
Early attempts(28) in 1925 by Shapiro and Neal and Hoff in 1942, and
Macus and Rambo(36) in 1955 failed to find an immunization treatment for
or an immune mechanism to explain cryptococcosis relative to the humoral
immune system.
Gadebush(14) in 1958, provided an interpretation of natural immunity
against Cj_ neoformans in rabbits which was accepted as valid. He intro
duced C . neoformans into rabbits through aerosols. Once C. neoformans
was situated in the alveolar septa and the alveoli of the lungs, the
majority was removed within 24 hours by neutrophils. However, the fungi
that were not phagocytosed by neutrophils proliferated freely. The phago
cytic obligation soon shifted to the monocyte. It is thought that during
this shift the fungi spread into areas outside the pulmonary tract(15).
After several days, the monocytes which initially confronted C_^ neoformans
matured into macrophages. They and other macrophages were activated by
delayed hypersensitivity reactions as both an effect of the cellular
immune response and through previous contact with the fungi as monocytes.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. -10-
i.e., direct contact activation. The existing C. neoformans were then
swiftly phagocytosed and destroyed. Complete recovery from this infection
was common if the infective neoformans dose was not so massive as to
overwhelm the host*s system.
Abrahams (1) in 1966 demonstrated that natural resistance to crypto
coccosis in mice was dependent on the cellular immune system and not the
humoral system directly. However, it was demonstrated that opsonization
of these fungi by IgG greatly enhanced phagocytosis. Abraham’s findings
were supported by Goren(17) in 1967.
Rabbit Macrophage; In previous study in our laboratory(11) it was
found that alveolar macrophages may be activated by either viable or non-
viable schenckii conidia regardless of the route of administration.
It was discovered that fungicidal activity was 2-to-3 fold greater in
those macrophages which had been recovered from animals sensitized by
aerosols than from those sensitized by intramuscular, intravenous, or
intraperitoneal injections of equivalent numbers of conidia.
S. schenckii Neutrophil Model: In 1973, Howard(23), using guinea
pig neutrophil cell cultures, demonstrated that neutrophils were highly
effective in killing extracellular Histoplasma capsulatum fungi cells.
Other studies (24) have also <^hown that neutrophils are highly effective
against certain extracellular fungi, such as Candida albicans, krusei,
parapsilosis, stellatordia, pseudotropicalis. Saccharomyces cerevisiate,
Geotrichium canidium, Aspergillus niger, fumigatus, and neoformans.
Macrophage Model: Howard(20), in 1959 maintained normal (noniramune)
mouse peritoneal exudate macrophage cultures inoculated with yeast phase
H. capsulatum. He found that as few as 30 fungus cells per macrophage
overwhelmed the macrophages.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. -11-
In 1965, Howard(21) expanded his previous studies dealing with
nonimmune mouse macrophages to other models. He used five different
isolates of capsulatum; he used both guinea pig and mouse normal
macrophages, and he used guinea pig and mouse macrophages in the pre
sence of specific hyperimmune serum and macrophage cells from specific
ally immunized guinea pigs and mice. He concluded that the generation
time of H^ capsulatum in mammalian macrophages was very constant, that
the rate of growth for the five 11^ capsulatum isolates was very similar
in macrophage cells; that the generation time of fungus within guinea
pig macrophages was nearly the same as in mouse macrophages, and that
the exposure of yeast phase H^ capsulatum to antibody and complement
did not affect intracellular fungal proliferation in the macrophages.
He reported no significant difference in fungus proliferation within
macrophage cells from immunized compared with those from nonimmunized
guinea pigs and mice.
In 1973 Howard(22) used freshly harvested macrophages from immunized
mice instead of those grown on tissue cultures. He found that H. capsulatum
was inhibited by these immune activated macrophage cells but was not killed.
In 1976 Howard and Otto(24) reported that the effectiveness of inhibit
ion of _H^ capsulatum was a product of the cellular immune system and not
the humoral system.
Kashkin et al.(27) in 1976, using the pathogenic fungi Coccidioides
immitis, the causative agent of the pulmonary infection coccidioidomycosis,
reported a similar finding that fungicidal and fungistatic responses are
dependent upon the cellular immune system which activated the peritoneal
macrophages. This is important to this author's research in that the
fungicidal and fungistatic effects of the cellular immune system against
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. —12—
S. schenckii shall be studied.
Statement of Purpose
This author proposes a slightly different approach In continuing
pathogenic fungal .research. The previously cited fungal models used
fungi that are not normally encountered by peritoneal exudate cells,
particularly peritoneal macrophages. capsulatum. C. neoformans. and
G. Immltls are basically pathogenic to the pulmonary system. To date
no study concerning a subcutaneous fungus/PEC model has been reported.
Hence, It Is felt that a guinea pig model using schenckii would be
of significance since peritoneal exudate cells do normally encounter
subcutaneous pathogenic fungi.
Therefore, the purpose of this thesis Is to determine some anti
fungal properties of guinea pig peritoneal exudate cells when chal
lenged by the pathogenic fungus, S_^ schenckii. the causative agent of
sporotrichosis, a lymphocutaneous Infection.
The first question to be evaluated Is: what phagocytic effects. If
any, do guinea pig peritoneal exudate cells have upon ^ schenckii?
The second question to be evaluated Is: given phagocytosis does
occur. Is It fungistatic or is It fungicidal?
The thlru questions to be evaluated Is: what are the effects of
cortisone compromise on PEC activity?
Plan of Development
In this study, ^ schenckii conidia will challenge thloglycolate-
Induced guinea pig peritoneal exudate cells In three specific modes:
normal. Immune, and Immune suppressed.
1. Normal condition: Defined as a guinea pig having had no previous
exposure to S. schenckii. This is also the control condition.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. -13-
2. Immunized condition: Defined as a guinea pig having been immu-
nized for both a primary and a secondary response against S. schenckii
antigens and therefore presumably capable of antigenic memory and macro
phage activation.
3. Immune suppressed condition: Defined as the state in which the
capacity to elicit the immune response has been greatly reduced by the
use of corticosteroids.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. —14-
CHAPTER II
MATERIALS AND METHODS
Animal Models
Guinea pigs, the animals models used in this research, were obtained
from Rocky Mountain Laboratories, Hamilton, Montana, were housed in the
animal room at the Department of Microbiology, were approximately the
same age, were immune competent, and were fed a nutritionally complete
commercial guinea pig ration.
Fungal Cultures
Cultures of ^ schenckii, strain 425, were obtained from stock cultures
stored at the Department of Microbiology, University of Montana. Subcul
tures of schenckii were inoculated onto Mycosel agar (Baltimore Biolo
gical Laboratory) slants; were incubated in screw cap tubes at 25°C, and
stored at minus 20°C. Before any S_^ schenckii culture was employed, the
culture was examined for contamination through microscopy and gross
observation. Broth cultures used for harvesting condia contained 150 ml
Sabouraud dextrose broth (Difco Laboratories) combined with 0.1% yeast
extract (Difco Laboratories). For conidia harvesting, a subculture of S.
schenckii was streaked on a Mycosel agar plate, which was incubated at 25°C
for 96 hours. After incubation, a 0.5 X 0.5 mm section of agar containing
S. schenckii was removed and inoculated into a 500 ml Erlenmeyer flask con
taining 150 ml of the conidia broth described above. Each inoculated flask
was then incubated at room temperature on a horizontal shaker for 10 to
12 days. At this time, the conidia were harvested by transferring about
75 ml of each broth culture into a 250 ml sterile plastic centrifuge bottle.
The bottles were centrifuged at 75 x G, at 4°C, for 10 minutes and the
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. — 15“
supernatant was discarded, leaving In each centrifuge bottle a pellet mass
of conidia. To each of the plastic centrifuge bottles, 100 ml of sterile
physiological saline (0.85%) were added. The bottles were hand agitated
25 times to Insure an adequate washing of the conidia. The conidia were
then recentrifuged as previously described and after centrifugation, a
second conidia washing was performed. These washed conidia were stored
In the plastic centrifuge bottles at minus 20°C for future use as either
cellular antigens or for use In establishing the effectiveness of PEC
phagocytosis In vitro.
Tissue Culturing Media
Eagles Minimum Essential Media (MEM) with Earles' balanced salt
solution (Microbiological Associates) was the tissue culture medium
employed. Two grams crystalline MEM were reconstituted In 1,000 ml triple
distilled water, buffered with 2 . 2 grams NaHCO^, and the hydrogen Ion
content was adjusted to pH 7.2 using 1 N NaOH or 1 N HCl depending upon
the acid or base concentration. This completed medium was filter steri
lized using a Seitz filter, pore size 0.22fim and stored at 4°C.
For the tissue culturing process, 100 ml MEM was enriched with 2%
heat Inactivated fetal calf serum (Microbiological Associates), 1 ml
trlclne (Sigma Chemical Co.) as a buffering agent, 10,000 units penicillin
G (Sigma Chemical Co.) and 10,000 pg streptomycin sulfate (Sigma Chemical
Co.) each to Inhibit gram positive and gram negative bacterial growth
respectively, 1 ml heparin (Sigma Chemical Co.) to prevent clumping of
red blood cells and 1 ml L-glutamlne (Sigma Chemical Co.) to ensure ade
quate levels of this amino acid. In this thesis, this solution shall be
known as complete MEM.
Phosphate Buffered Saline (PBS) was utilized as a physiological buffer-
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. —16—
ing agent and in the tissue culture washing process to be described later.
Stock solutions of PBS were prepared as described by Dulbecco and Vozt(9).
Glassware
All glass items utilized in this work were first rinsed in cool water
to avoid protein coagulation, then washed in warm soapy water, rinsed 5
times in distilled water, and finally acid rinsed for a minimum of 4 hours
in a 0.1 N HCl immersion bath. In cases where direct contact between PECs
and glassware occurred, those glasswares were siliconized with Silaclad
(Biological Associates) with the exception of the 15 mm-diameter cover
slips positioned in the bottom of each culture well in order to augment
PEC adherence.
Antigen Preparations
Two types of cellular antigens were used in this work; viable and
non-viable S. schenckii. (1) Viable S. schenckii; As required, samples
of schenckii conidia were transferred from the 250-ml plastic centri
fuge bottles, plated on Mycosel agar, incubated at 25°C, and inspected
for growth on the 5th day of incubation in order to ensure viability.
Preparations of viable conidia were produced by adding conidia to sterile
physiological saline until a concentration of 1 0 ^ conidia per ml saline
was achieved. These viable conidia preparations were stored at 4°C in
screw cap bottles. (2) Non-viable S. schenckii: S. schenckii viable
conidia were added to a 1 :1 0 , 0 0 0 concentration of merthiolate, incubated
at 35°C for 24 hours and washed in PBS to remove the merthiolate. This was
accomplished by centrifuging the merthiolate aliquot for 15 minutes at 4®C,
at 75 X G. Once completed, the merthiolate supernatant was decanted
followed by the addition of 50 ml PBS. This mixture was hand agitated 25
times to ensure adequate washing, followed by recentrifugation as previously
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. -17-
described. At this time, a sample of the conidia from this washed
aliquot was plated on Mycosel agar, incubated at 25°C, and inspected
for growth after 72 hours. Non-viability was validated by the absence
of any conidia growth when plated on Mycosel agar. Non-viable conidia
were also stored at a 1 0 ^ conidia per ml physiological saline concen
tration at 4°C in screw cap bottles.
Experimental Models
Group I: This, the control group, received neither an immunization
of fungal conidial antigen or cortisone.
Group II; This group was immunized with merthiolate-treated, non-
viable S_^ schenckiL conidia. The guinea pigs in this group were initially
immunized intraperitoneally at day 1 with a 1 0 ^ conidia dose followed by
a second 10^ conidia dose at day 11, and the PECs were extraced at day 22.
Group III: The animals in this group were immunized with schenckii
viable conidia. Each animal received two 10^ doses of viable conidia, the
first at day 1 and the second at day 1 1 , administered intraperitoneally.
PECs were collected at day 22.
Group IV: The guinea pigs in this group were administered hydro
cortisone acetate (Sigma Chemical Co.) but no fungal antigen. Each animal
received 0.05 mg hydio-cortisone acetate (cortisone) per gram body weight
intraperitoneally at 5-day intervals until day 22. At this time the PECs
were removed.
Group V: This group was treated with cortisone and immunized with
non-viable _S^ schenckii conidia. Two days prior to the first 10^ non-
viable conidia immunization, 0.05 mg cortisone per gram body weight was
injected intraperitoneally into each guinea pig; the injection was repeated
at 5-day intervals until day 22. Non-viable conidia in a 10^ dose were
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. —18—
injected into each animal at days 1 and 1 1 , followed at day 22 with the
collection of PECs.
Group VI: This group was injected with cortisone and immunized with
viable ^ Schenckii conidia. Guinea pigs received 0.05 mg dose of cortisone
per gram body weight intraperitoneally 2 days prior to the first immunization
of 10^ viable conidia. Cortisone was readministered at 5-day intervals
thereafter until day 22. At days 1 and 11, non-viable conidia were
injected into each guinea pig intraperitoneally and at day 22 the PECs were
collected.
It should be mentioned that in each of these 6 models, 72 hours prior
to removal of the PECs (or on day 19), each guinea pig was induced with an
injection of 4 ml of thioglycolate fluid medium (Difco Laboratories) intra
peritoneally.
In Vitro Methodology
In order to study the in vitro effects of PECs when exposed to S.
schenckii conidia, PECs were introduced to viable conidia through a tissue
culturing procedure.
Procedure
1. At day 22, 20 ml complete MEM was injected intraperitoneally via
an 18 gauge needle into each guinea pig. This was followed by a gentle
massage of the abdominal area for approximately 1 minute to increase the
number of PECs collected. At this time the needle was removed from its
syringe and replaced, bevel up, into the initial puncture wound at an acute
angle of 20-40° from the plane of the guinea pig's abdomen until it was in
the peritoneal cavity. A prechilled 40-ml sterile siliconized centrifuge
tube was held under the needle's butt to collect the returning PEC suspension.
To ensure the maximum recovery of the PEC suspension, a gentle downward
abdomen milking was provided each animal.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. —19—
2. After the PEC/MEM aspirates were collected from each of the six
guinea pig models, they were centrifuged 5 minutes, at 4°C, at 75 X G,
in the 40-ml centrifuge tubes.
3. Once centrifugation was completed, the supernatant was aspirated,
leaving a pellet of PECs at the bottom of each of the tubes. At this point
1 ml 9.17 M Tris-(hydroxymethyl)-aminomethane (Sigma Chemical Co.) and 9 ml
0.83% NH^Cl were added to each centrifuge tube containing the PECs. This
suspension was allowed to stand at room temperature for 15 minutes to en
sure lysis of any red blood cells collected.
4. The cells were then re-centrifuged as in step 2 followed by aspi
ration of the supernatant fluid. Once completed, 10 ml PBS were added to
each centrifuge tube to wash the PECs, followed by centrifugation as in
step 2 .
5. At this point, the supernatant was aspirated and the PECs were
resuspended in 5 ml of complete MEM per each centrifuge tube. A small
volume of PEC/MEM suspension from each tube was collected and placed on
a hemocytometer. The PECs were then counted by standard methods(4) and
these counts estimated the number of PECs per ml within the tubes.
Complete MEM was added to each of the 40-ml centrifuge tubes until a 10^
PEC count per ml was confirmed via counting.
6 . After a uniform cell count of 10^ cells per ml was achieved for
each of the 6 models, 1 ml PEC aliquots were added to each of the 24, 1.7
X 1.6 cm wells contained in each flat bottom tissue culture plate (Flow
Laboratories). Each well in tissue culture plates contained a round 15
mm flat non-siliconized glass cover slip for PEC adherence.
7. These tissue culture plates were incubated at 37°C in 0.4% CO^
for 45 minutes. At this time any non-adherent PECs within each culture
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. —20-
well were removed by dispensing 5 ml PBS into each well simultaneously
removing the PBS/MEM solution through a Pasteur pipette via negative
pressure. To prevent the PECs from osmotic shock, approximately a 2 mm
layer of PBS covering the adherent cells on the coverslips was not
removed. Quickly, 1 ml of MEM was reintroduced into each of the tissue
culture wells. Once completed, the PEC tissue culture plates were re
incubated. Refeeding was performed every 4 hours in an identical manner
until the PECs had been cultured for a total of 12 hours. It was found
that 12 hours incubation was necessary for an even monolayer of PECs to
form on each of the cover slips.
8 . At hour 12, each of the tissue culture wells was washed with 5
ml PBC as stated in step 2, followed by the introduction of 10“^ viable
S. schenckii conidia suspended in 1 ml of complete MEM.
9. At times 0, h, %, 1, 2, 4, 8 , and 12 hours, a cover slip con
taining adherent PECs, representing each of the 6 models, was removed
from the appropriate tissue culture well, washed in physiological (0.85%)
saline, and stained with a 1 :1 0 , 0 0 0 dilution of acridine orange in a
modified procedure described by Newcomer(40). Following the acridine
orange staining method, conidia which are viable appear bright green while
conidia which are non-viable appear orange or red when examined with fluo
rescence microscopy.
10. Two methods of conidia counting were employed. In the first
method, 20 PECs, selected at random, were observed microscopically. The
number of conidia contained within each of these 2 0 phagocytes was tabu
lated and averaged. The second counting method used the same data as the
first except any of the PECs not actually demonstrating phagocytoses via
the acridine staining method were dropped from the tally and an adjusted
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. -21-
average number of conidia phagocytosed per PEC was calculated. This was
performed in order to allow for the possibility that the PECs which did
not demonstrate intracellular conidia may have been non-viable PECs
instead of viable, non-phagocytosing PECs.
Quantitative Analysis
The Statistical Package for Social Science (SPSS) computer program
format was employed on the University of Montana’s DEC-20 computer system
when possible. When hypothesis testing was encountered, the 0.05 level
of significance (95% confidence) for type I error was used. When appro
priate, the descriptive level of significance or P value is given. This
is defined as the probability of realizing an observed value or a more
extreme value, given that the null hypothesis of equivalence amount models
is true.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. - 22-
CHAPTER III
RESULTS
Plotted on figure 1 are the average numbers of phagocytosed conidia
per PEC, for each of the 6 models based on microscopic observations of
20 randomly selected PECs per model, plotted vs time in hours. Note that
the time intervals from time zero, when viable conidia were first intro
duced into each tissue culture plate wells, to the first observed cyto
toxic effects of these PECs on the conidia are also provided. Table 1
provides the mean data of these observations from which graph 1 was plotted.
Figure 2 provides the plot of the average number of phagocytosed
conidia for each guinea pig model relative to time; however, this graph
portrays only those PECs having actually demonstrated phagocytosis of
conidia. Those PECs having not phagocytosed any conidia as demonstrated
microscopically by means of the acridine staining procedure, were dropped
from this statistical design. Table 2 provides the mean data from which
Figure 2 was plotted.
A standard 1-factor, 2-way analysis of variance(46), Table 3, was
computed using the mean data contained in Tables 1 and 2 and other
required parameters, for example, che errors of the means, sums of squares,
F statistics and variances of these sample data necessary in this Anova
design were computed from the raw experimental data. Computational analysis
was performed through an ANOVA SPSS computer program(41), followed by both
the computation of 4 multiple contrasts and hypotheses testing by this author,
using a hand-held programmable HP-41C calculator. Since the same relative
statistical results were achieved when using the data from either Table 1
or 2 , (as confirmed through a non-pararoetric statistical process involving
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. "OCD O Q. C g Q.
"O CD
C /) f u n g i c i d a l 3o' 0 5 EFFECT FIRST NOTICED CD 8 CONIDIA
(O'3" 1 3 CD
3. 3" I CD Ni "OCD O Q.C a o 3 ■D O
CD Q.
■D CD 0 1/4 1/2 1 2 4 6 8 10 C /) TIME IN HOURS o' 3 VIABLE CONIDIA 1--- 1— CORTISONE COMP, WITH VIABLE CONIDIA
NON-VIABLE CONIDIA — O — O - CORTISONE COMP, WITH NON-VIABLE CONIDIA CONTROL “t A Ùr- CORTISONE COMP, WITH NO IMMUNIZATION
FIGURE 1. AVERAGE NUMBER OF CONIDIA PHAGOCYTOSED PER PEC BASED OK 20 RANDOMLY SELECTED PECs. 73 ■DCD O Q. C g Q.
■D CD
C /) 3o' O X « FUNGICIDAL
EFFECT FIRST NOTICED 8 CONIDIA
i
3. 3" CD
CD ■D I O N) Q. •C- C a t O 3 ■D O
CD Û.
■D CD i (gC /) 0 1/4 1/2 1 o"
TIME IN HOURS
VIABLE CONIDIA 4— CORTISONE COMP, WITH VIABLE CONIDIA NON-VIABLE CONIDIA CORTISONE COMP, WITH NON-VIABLE CONIDIA ^ CONTROL CORTISONE COMP, WITH NO IMMUNIZATION
FIGURE 2. AVERAGE NUMBER OF CONIDIA PHAGOCYTOSED PER PEC BASED ON PECs CONTAINING CONIDIA ONLY ■DCD O Q. C g Q. TABLE X
■D CD
CW /) Average number of conidia phagocytosed per PEC based on 20 randomly selected PECs, 3o" 0 3 CD Animal Treatment Schedule 8 Cortisone Cortisone comp., ci' 3" Viable Non-viable Normal comp,, plus plus non-viable Cortisone 1 Time (hr) conidia conidia control viable conidia conidia control 3 CD
3. 0 0 0 0 0 0 0 3" CD 1/4 1 0.5 0.5 0 0 0 ■DCD O Q. C 1/2 1 1 0.5 0 0 0 a O 3 ■D 1 2 1.5 1 1 0.5 0 O 2 8 2 3 3 1.5 1 CD Q. 4 16 4 3 3 2 1
■D 6 20 4 3 4 2 1 CD
C/) C/) 8 20 5 4 4.5 2.5 1.5
12 20 6 5 5 3 2 ■OCD O Q. C TABLE 2 g Q.
■o CD Average number of conidia phagocytosed per PEC based on only those PECs containing conidia, WC /) 3o" 0 3 CD Animal Treatment Schedule 8 Cortisone Cortisone comp,, ci' 3" Viable Non-viable Normal comp., plus plus non-viable Cortisone 1 Time (hr) conidia conidia control viable conidia conidia control 3 CD "n c 3. 0 0 0 0 0 0 0 3" CD
CD 1/4 1 0.5 0.5 0 0 0 ■O O Q. C 1/2 2 1 1 0 0 0 a O 3 ■O 1 3 2 2 1 1 0 O 2 10 5 5 4 2 1 CD Q. 4 18 6 5 5 3 2
■O 6 20 6 5 5 3 2 CD
C/) C/) 8 20 7 6 5 3 2
12 20 8 7 7 4 3 ■o I ANOVA summary table of the average number of conidia phagocytosed per 20 randomly selected PECs. I Variation Degrees Sum of Source______Freedom______Squares_____ Mean Squares______F Computed______F Tabled %
C /) Due treatments j 696.3 139.26 3.29 2.13 CO o'3 o Due time 7 296.3 42.33 5.21 1.57
CD 8 Residual (unex plained by ANOVA) 12 97.5 8.12 - c5' o 3 CD
C p. ANOVA summary table of conidia phagocytosed per PEC based only on those PECs containing conidia
■oCD O CQ. Variation Degrees Sum of a o Source Freedom Squares Mean Squares F Computed F Tabled 3 ■a o Due treatments 5 693.8 138.76 3.32 2.13
Q.CD Due time 7 292.5 41.79 5.16 1.57
Residual (unex ■D plained by ANOVA) 12 97.2 8.10 CD 3 CO CO o'
Variation Due to Treatments = SSa = (Vi — v)
Variation Due to Time = % » Residual or Unexplained Variation = -/j ■o o Q. C Summary table of the nested ANOVA design based on phagocytic efficiency on time g Q.
■o CD
C /) C /)
Source of Sum of Degrees Variation Squares Freedom Mean Square F computed F tabled 8 3 c5' Due treatments 1583.51 316.701 11.54 4.26
Within treatment among PECs 3 I 3" on time 576.11 21 27.434 12.75 2.04 CD Ni
CD ? ■o Residual O Q. C a (random error) 1032.95 480 2.152 3o "O o
CD Q. Variation Due Treatments = "x) A*l TD Variation Within Treatments CD Contrasts among PECs, with % ' b" / — ‘ C/) time in the model = trv? C/) A*t Residual (unexplained error) = ? ? E ( v ■DCD O Q. C 8 Q. TABLE 5
■D CD
C /) ANOVA Summary table for average numbers of conidia phagocytosed for each model regressed C /) on (X^) treatment, and contrasted with the addition (X^) Age, and/or (X^) Sex.
Variation Degrees Sum of Square F computed Model Source Freedom Variation
^Xj^ due treatment 582.95 19.67
%2/Xl due age with 3 treatment in the 3" model, but controlled 0.24 0.08 CD Variation explained CD T3 by multi / X]/Xi due sex with O Q. ple re treatment in the I C gression model, but controlled 0.03 0.001 % a I 3o of X^, X?, T3 X] on Y*" X^/Xi.Xj due treatment O with age and sex in . the model, but CD Q. controlled 1 0.29 0.11 r
Unexplained Variation 8 195.19 22.69
■o CD
(/)C/) F computed * é - i h É i i ^ Æ — i-e' % Y = number of conidia phagocytosed
* computed via an SPSS Algorythnn -30-
a modified Chi Square procedure(38) measuring a goodness of fit which
provided a P value of 0.005, which is the probability of a significant
difference between the average data contained in Tables 1 and 2, which
is insignificant), only those data based solely on those PECs containing
intracellular conidia (Table 2) will be used throughout the remainder of
this work.
Based on the 2-factor 1-way ANOVA using the data on Figure 2, two
separate groups of models could be isolated: the first group, the model
immunized with viable conidia; and the second group, which contained the
other 5 models, which as a unit, were less efficient in antifungal
properties. It can be seen that the PECs extracted from guinea pigs immu
nized with viable conidia were the most effective of those models tested,
in both their phagocytosing and their fungicidal activity against schen
ckii. The probability of this group not being the most efficient and active
phagocytic group studied is less than 0.001. This group phagocytosed an
average of more than 20 conidia per PEC within the 8 hour time frame and
that intracellular counting of PECs had to be terminated at hour 8 ,
because the numbers of intracellular conidia could no longer be accurately
counted, since the PECs were literally "stuffed" with intracellular conidia.
The phagocytic rate between hours 1 and 4 was about 7 conidia per hour,
which was more than double the average rate of any of the other 5 groups.
Intracellular conidia lost viability less than 2 hours after initial
exposure to PECs in this group as demonstrated by the acridine orange
staining procedure.
The non-cortisone compromised animals immunized with non-viable conidia
and the control group were statistically comparable in both fungicidal effects
and phagocytic effects, phagocytosing an average of 8 - 7 conidia per PEC
respectively within 12 hours. The first fungicidal effects against the
conidia appeared about 4 hours after initial exposure to the PECs.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. —31—
Neither phagocytic nor fungicidal activity of PECs from any of the
cortisone compromised animals sensitized with viable conidia differed
significantly from the normal untreated, unsensitized animals. The phago
cytic efficiency of PECs from the compromised animals challenged with
non-viable conidia was minimal and did not differ significantly from the
unchallenged animals. The first fungicidal effects against the conidia
appeared 8 hours after initial exposure to the PECs for each of the 3
compromised groups.
Using only one animal from each model for each experimental run
attributed to large internal statistical variations, causing loss in
quantitative precision via the 1-way, 2-factor ANOVA procedure. To
achieve a greater degree of quantitative resolution through statistical
analysis, a nested ANOVA design(lO) was programmed into the DEC-20
computer system, using the SPSS format. Unlike the 1-way, 2-factor
ANOVA design, which contrasted the variation of the 6 guinea pig models
to each other based on time, the nested ANOVA design provided a 3-dimen
sional test by comparing the variation among the 6 guinea pig models based
on time with the additional subsample contrast, that of phagocytic varia
tion among PECs, both within each of the 6 models (internal variation)
and between each of the 6 guinea pig models. Since a statistical sampling
procedure had been violated using the nested ANOVA design in that for each
model, several guinea pigs within that model should have their extracted
PECs pooled, but did not, the P value is significantly high (the probabi
lity of this nested ANOVA design being untrue is P=0.30), which provided a
higher probability of being incorrect than the 2 factor 1 way ANOVA of
P 0.001 does.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. —32—
Via the nested design, 3 distinct animal groups, based on phagocytic
efficiency, could be realized using multiple contrast analyses based on
the data contained in the Nested ANOVA Summary table. Table 4. The most
obvious difference between the 3 groups appeared in the animal model immu
nized with viable conidia. Within the other 2 models, separated via the
nested design, was the second most efficient phagocytic group consisting
of 3 models: the normal animal model immunized with non-viable conidia,
the normal control model, and the cortisone compromised model immunized
with viable conidia. These three models were statistically identical at
the 95% confidence level based on their similar phagocytic efficiency.
The third group isolated and the least efficient of the 3 models included
the two remaining cortisone compromised models, those animals immunized
with non-viable conidia and the cortisone compromised control animals.
The probability of this third group being statistically identical to the
second class in phagocytic efficiency is 0.30.
Of concern in this work was the possible bias effects that both
differences in sex and age might have in this study. Prior to actually
initiating any of the phagocytic and fungicidal assays as described in
this thesis, a multiple/partial and a partial regression analysis were
performed using the SPSS and BASIC programming formats. .Four experimental
contrasts were devised using a 2 X 2 factorial format similar to those
discussed by Cochran and Cox(6 ) so as to ensure that each animal model
was both age and sex adjusted. That is, each model was randomly assigned
1 of 4 variables: an older guinea pig, a younger guinea pig, a female guinea
pig, and a male guinea pig. Each of these variables was then used one time
within each model during the 4 experimental runs used in completing this
study. Internal variation within each model was then measured and a
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. -33—
Partial F Test ANOVA procedure(28) was employed (Table 5), measuring
first the variation explained solely by phagocytic variation due the 6
treatments which was high (F computed = 19.67). Next the variable,
sex, was measured to find how much additional phagocytic variation
could be explained through this influence. As can be seen on Table 5,
the additional phagocytic variation explained by sex is negligible (F
computed -> 0.08). Next the sex variable was removed and the guinea pig
factor tested. The amount of phagocytic variation explained solely by
age was also negligible (F computed = 0.03). Finally, both variables,
age and sex, were simultaneously measured to find their joint contribut
ion to explaining phagocytic variation. Again, the contribution in
explaining additional phagocytic variation was negligible (F computed =
0.11).
Hence, it can be concluded that sex and the age differences in this
work did not bias the phagocytic results at a probability level of con
fidence of 0.999.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. — 34-
c h a p t e r IV
DISCUSSION AND CONCLUSIONS
The immunological mechanisms of resistance to the pathogenic fungus
S. schenckii are not fully understood. According to the literature, the
PEC/fungus relationship is similar to the PEC/bacterium relationship, in
that when conidia or bacteria are encountered in a wound such as a trau
matic implantation within cutaneous tissues, neutrophils play a paramount
antimicrobial role since they are physically present at the wound site
from the onset of the inflammatory process. Since neutrophils require
no activation to initiate efficient phagocytosis, this phagocytic res
ponse occurs whether or not the animal has been previously immunized
against that foreign body. In both human mycoses and bacterial infect
ions, neutrophils soon become of lesser phagocytic importance against
those organisms, relative to the activated macrophage, once T-cell secre
tion of lymphokines such as MAP and MIF has occured mainly due to the
lower numbers of neutrophils but higher number of activated macrophages
found in cutaneous tissue. Also, the physically smaller neutrophil
simply is not capable of phagocytosing the large number of microbial
organisms that the macrophage can.
However, a significant post-phagocytic difference between the bac
terial and fungal models may exist. Recall that once a bacterial or
fungal cell comes in physical contact with a neutrophil, pseudopodia
from this white blood cell extend and surround the organism, encasing it
within a portion of inverted plasma membrane, the phagosome. Once the
organism is encased in the phagosome, the entire body, assisted by micro
tubules, moves toward lysosomes(25). Once the phagosome and lysosomes
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. - 35-
come in physical contact, they fuse, exposing the organism to an environ
ment of low pH, myloperoxidase, hydrogen peroxide, lactoferrin, glyco-
sidases, proteases, lipases, granular cationic proteins, and a halide,
usually iodide. For the majority of bacteria, the fate is degradation
but this may not be the case with fungi. Fungi have cell wall structures
significantly different biochemically from either gram negative or posi
tive bacteria. The fungal cell wall consists mainly of polysaccharide
polymers of cellulose, of chitin, of beta-glucans, and of mannans, either
in combination with each other or appearing as single repeating polymers.
Mammalian PECs are known to lack enzymes capable of degrading these
unique polysaccharide configurations, even after intracellular killing
of these fungi has been demonstrated(7). Therefore, the retention of
fungal bodies may persist intracellularly. This may play a significant
role in human resistance to fungal infections since most of the non-
degradable fungal cells are partially degraded by phagocytes. This
partial degradation, however, is at best, slow, hence it may provide an
immunological "boosting" effect by intensifying the immune response.
Some disagreement exists as to whether non-viable fungal antigens
are as immunogenic to a mammalian host as viable fungal antigens. Howard
(19) reported that immune peritoneal macrophages in both mice and guinea
pigs were activated to a greater extent when immunized with viable H.
capsulatum than with non-viable H. capsulaturn conidia. Others (13,14,47)
have reported similar findings. However, using rabbit models, Ferguson
(1 1 ) reported that activation of alveolar macrophages was relatively con
stant regardless of whether viable or non-viable S_^ schenckii were admini
stered as antigens. Using bacterial models, Mackaness(34) reported that
viable L. monocytogenes were more antigenic than non-viable L . monocytogenes.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. -36-
as did others(2>36,37) using various other bacterial models. It appears
then that both viable fungi and viable bacteria are generally more
antigenic than non-viable types. From the results of this present work,
it has been demonstrated that viable conidia are more highly antigenic
than non-viable S . schenckii conidia when administered into guinea pigs.
Both the phagocytic rate and the numbers of conidia phagocytosed by PECs of
normal animals immunized with viable conidia were at least twice that of
the PECs from animals immunized with non-viable conidia. Also, from these
results, it can be seen that both those animals immunized with non-viable
conidia as well as the non-immunized controls were equivalent in phagocy-
totic ability, which probably means that immunization with non-viable
conidia has no detectable effect on the immune system in terms of PEC
activation. In the present study, the results obtained through the 2-
factor, 1-way ANOVA statistic, indicate that these 6 guinea pig models
could be separated into 2 groups based on their phagocytic effectiveness.
The most efficient animals in phagocytotic ability were those immunized
with viable conidia. The other 5 models were statistically indistinguish
able from each other. The conclusions reached using this statistical
analysis are probably not complete in describing the phagocytic variation
amoung the 5 models contained in group 2 , since it may corsist of 2 separate
subclasses. With a more sensitive nested ANOVA design, it was determined
that 3 separate classes may exist within the 6 models. The most effective
phagocytic group consisted of those animals immunized with viable conidia.
The second most effective group consisted of 3 animal models: control
animals immunized with non-viable conidia; animals compromised with cortisone
and immunized with viable conidia; and animals immunized with non-viable
conidia. These models were equivalent in phagocytic efficiency. The third
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. -37-
and least effective group isolated by this statistical process was
composed of the two cortisone-compromised animal models, those immunized
with non-viable conidia and the non-immunized controls.
Unfortunately this nested design is not above reproach as protrayed
by the relatively low (0.70) probability of being the correct model of
representation, because the requirement for pooling guinea pig PEC samples
within each of the 6 models was not met: too large a number of guinea
pigs would be required for such a test. However, the 3 phagocytic group
models appear logical and may be quite valid.
Note that the normal controls and the cortisone-compromised models,
both those immunized with viable conidia and those non-immunized, are
equivalent in phagocytotic efficiency. Cortisone is known to prevent the
fusion of phagosomes and lysosomes(16) which leads to the inhibition of
enzymatic degradation and alters the plasmalemma inhibiting phagocytosis.
It is also known that cortisone is lymphotoxic and may lead to reduction
in the number of T lymphocytes which are required in PEC activation, as
well as to increased release of neutrophils from the bone marrow into
peripheral blood pools.
It is possible that the cortisone-compromised anamals which were
immunized with viable conidia demonstrated PEC activity equivalent to
the non-immunized control group because immunization with highly antige
nic viable conidia may have compensated for the immune-compromising
effect of cortisone treatments.
Examining the last group, those models cortisone-compromised and
immunized with non-viable conidia and the non-immunized cortisone-
compromised group, it can be argued that there was a more dramatic
repression of the immune response, because this group, based on the
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. —38—
nested ANOVA design, was least efficient in phagocytosis than the other
model groups.
Using lower levels of cortisone and/or different injection inter
vals might have produced other results. For example, it was discovered
that a dose of 0.005 mg cortisone per gram animal weight had little
repressive response on the guinea pig immune system, nor did a 0.05 mg
per gram body weight cortisone injection, administered in 3 divided
doses at ten-day intervals. Conversely, it was found that a 0.05 mg
cortisone per gram body weight injection administered in 7 divided doses
at 2-day intervals killed all guines pigs receiving that treatment, per
haps because the animals lost all immunological resistance or suffered
toxic effects of the drug. Hence the 0.05 mg cortisone per gram body
weight administered in 5 divided doses was found most suitable for this
work.
A significant point in this research was the association between
PEG activation states and cytotoxic activity. One can see (Fig. 1) that
fungicidal effects occurred with those conidia phagocytosed by the non
cortisone compromised animals and immunized with viable conidia within
half the time required for fungicidal effects to be first noticed in the
non-cortisone compromised animals immunized with non-viable conidia and
the normal controls. This observation has not previously been reported.
One would, of course, expect the fungicidal effects of the cortisone-
treated animals to be reduced for reasons stated above.
Controversy exists as to the role of the humoral immune system in
relationship to mycotic disease. Recall that antibodies are produced
in most mycotic infections. Their production of course depends in part
on actual physical contact between fungi and/or fungal metabolites and/
or B lymphocyte recognition of those antigens.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. -39-
In the case of acute respiratory infection as in histoplasmosis,
(caused by capsulatum) detectable IgG, IgM, and IgA immunoglobulins
corresponding to capsulatum protein and polysaccharide antigens can
be demonstrated from 10-14 days after the initial exposure to the fungus.
Medically, to some extent, the detection of serum antibodies is useful
in determing the status and progress of deep fungal infections, including
human sporotrichosis. But evidence for direct antibody killing of fungi
is scarce. Ferguson(ll) however, reported that schenckii conidial
growth was inhibited by incubating conidia in the presence of antiserum.
Lurie and others(31,33) have demonstrated that S . schenckii conidia and/or
yeast-phase cells are inhibited within the "asteroid body", an eosinophilic
immune complex precipitate occurring in histological sections from sporo-
trichic lesions, and may be converted into "chlamydospores" or (sclerotic
bodies), i.e., thick-walled, non-proliferating cells, in the presence of
increasing immunoglobulin titers. Also, it has been reported that C.
neoformans has been killed in the presence of anti-cryptococcal antibody.
Also that the C-3 component of complement when activated by ^ albicans,
via the alternate pathway, has been responsible for direct killing of this
fungus. Others state that beneficial effects of antibody relative to the
host against fungi are inconclusive mainly due to lack of serious study.
Aside from any fungicidal effects immunoglobulins may have upon
fungi, the ability of PECs to phagocytose fungi may be enhanced when
immunoglobulins coat fungal cells opsonizing them for PECs, augmenting
phagocytosis.
It is commonly observed in clinical settings that only low numbers
of fungal elements are normally seen in exudates, tissues, and aspirates
from humans infected with S. schenckii. Given that human lymphatic phago
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. -40-
cytes and interstitial cutaneous histiocytes are approximately equal to
the guinea pig PECs in phagocytic effectiveness, the result of the
present work may offer insight into this phenomenon. Since S. schenckii
antigens are probably ubiquitous in nature, especially on wood products,
it is likely that humans are normally exposed to at least low numbers of
these antigens. That exposure can often be confirmed via positive skin
tests of persons never having had demonstrable sporotrichosis. It stands
to reason, then, that immunological memory is present to elicit the
immune response when S. schenckii is encountered in a wound. The PECs,
of course activated in part by T memory cells, would be highly efficient
in fungal cell killing. In fact, the observation of low numbers of fungal
elements seen in fixed cutaneous and other localized atypical sporotrichic
infections may be indicative of even more effective activation of human
interstitial histiocytes. The results of Ferguson(11) indicate that this
concept may reach maxium significance in the alveolar macrophage which are
generally so efficient in conidial killing that pulmonary disease rarely
occurs in the normal individual.
In the presence of demonstrable antifungal phagocytic activity as
portrayed in this work (or equivalently in humans, the interstitial and
paralymphatic phagocytes) th: chronicity of sporotrichosis in the presence
of highly efficient phagocytes may be based on the low numbers of normal
or activated phagocytes present in tissues to remove schenckii com
pletely, and/or the failure of some phagocytes to be activated, possibly
because of reduced immune response to schenckii antigens actually
present in the infection process due to fungal elements being walled off
in granulomatous lesions and the formation of non-phagocytosing giant cells
by macrophage fusion.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. -41-
In sporotrichosis, as caused by traumatic Implantation of S_^ schenckii
Into cutaneous and lymphocutaneous tissues, after about 3 weeks from the
Initial time of fungal Inplantation, a small, hard movable, nontender nodule
appears. Histological sections reveal this to be a granulomatous lesion
containing fungal debris, walled off by phagocytes, elastic fibers, coll
agenous fibers, and fibrocytes. This walling off of fungal cells may prevent
B cells from recognizing fungal surface antigens and T cells from receiving
processed Internal, partially degraded, fungal antigens from giant cells,
thus Impairing the Immune response. In chronic sporotrichic Infections
where the fungal elements multiply faster than they can be phagocytosed,
however, they disseminate through lymphatic vessels, which are normally
sparse In phagocyte numbers, so fungal proliferation may not be Inhibited.
Once these fungi have spread to a lymph node, the majority of fungi sus
pended In the lymph are removed by filtration and phagocytosed by both
granulocytes and monocytes. Those fungal elements escaping phagocytosis
are free to proliferate Into the subcutaneous tissue and renew this pro
cess over and over until a regional lymph node Is encountered. Rarely will
S. schenckii escape complete phagocytosis once contained In one of these
nodes.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. -42-
CHAPTER V
SUMMARY
Six guinea pig models were treated with ^ schenckii conidia in
various combinations to ascertain the effects of peritoneal exudate cells
(PECs) on these conidia, to find whether or not those PEC effects were
fungicidal or fungistatic to S_^ schenckii conidia, and to determine the
effects cortisone compromise had upon PEC activity. Phagocytic responses
were measured using these 6 animal models which included animals immu
nized with viable conidia, animals immunized with non-viable conidia,
untreated controls, animals compromised with hydro-cortisone acetate but
not immunized, animals both compromised and immunized with viable conidia
and finally animals both compromised and immunized with non-viable conidia.
Statistical analyses were used to compare the groups. It appears that
animals immunized with viable conidia are more efficient in clearing these
fungi via phagocytosis, that phagocytic rate and fungicidal properties are
positively correlated, and that cortisone compromise, achieved through
repeated cortisone injections into these animals, reduces both phagocytic
ability and fungicidal properties of PECs.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. —43-
REFERENCES CITED
1. Abrahams, I. 1966. Further studies on murine cryptococcosis. J. Immunol. 96: 525,
2. Allison, A. C. 1974. On the roll of mononuclear phagocytosis in immunity against viruses. Med. Virol. 1^: 15-17.
3. Blanden, R. V., Mackaness, G. B., and Collins, F. M. 1966. Mechanisms of acquired resistance in mouse typhoid, J. Exp. Med. 124: 585-600,
4. Bryant, N. 1979. Laboratory Immunology and Serology, pp 3-32. Saunders, Philadelphia.
5. Carpenter, P. L. 1975. Immunology and Serology. 3rd Ed. p 8 . W. B. Saunders, Philadelphia.
6 . Cochran W. and Cox, G. 1957. Experimental Design. 2nd Ed. pp: 102-156. John Wiley, New York.
7. Dick, A. 1980. Infection and Disease, pp: 89-148. Thornbriar Press, Calif.
8 . Dougherty, W. M. 1976. Leukopoiesis, particularly of the neutrophil. Introduction to Hematology, pp: 27-95, 2nd Ed. Mosby, St. Louis.
9. Dulbecco, R., and Vozt, A., 1954. Experiments in tissue cul turing, J. Exp. Med. 92: 167-168.
10. Dunn, O., and Clark, V. 1974. Applied Statistics: Analysis of Variance and Regression, pp: 96-118, John Wiley and Sons, New York.
11. Ferguson, R. 19^8 Immune Responses in Rabbits to Sporothrix schenckii. Master’s Thesis, University of Montana.
12. Fudenberg, H. H., Stites, D. P., Wells, J. V. 1978. Basic and Clinical Immunology. 2nd Ed. pp: 219-228. Lange, Los Altos, Calif.
13. Fudenberg, H. H., Stites, D. P., Wells, J. V. 1978. Basic and Clinical Immunology. 2nd Ed. pp: 96-108. Lange, Los Altos, Calif.
14. Gadebush, H. H. 1958. Active immunization against C. neofor- mans. J. Infect. Dis. 102: 217-219.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. -44-
15. Gadebush, H. H., and Gikas, P. W. 1965. Effects of cortisone in pulmonary cryptococcosis. Amer. Rev. Resp. Dis. 92i 64-69.
16. Goodman. L., and Gilman, A. 1975. The Pharmacological Basis of Therapeutics. 5th Ed. pp: 1472-1503. Macmillan, New York.
17. Goren, M. B. 1967. Experiments in murine cryptococcosis. J. Immunol. ^ 8 : 914-921.
18. Hart, D., and Armstrong, J. 1974. Strain virulence and lysosomal response in macrophages infected with Mycobacterium tuber culosis . Infec. Immun. 10: 742-746.
19. Hirsch, M. S., Sisman, B., and Allison, A. C. 1970. Macrophages and age dependent resistance to Herpes Simplex virus in mice. J. Immunol. 104: 1159-1160.
20. Howard, D. H. 1959. Observations on tissue cultures of mouse peritoneal exudates inoculated with H^ capsulatum. J. Bacteriol % 8 : 69-77.
21. Howard, D. H. 1965. Intracellular growth of H. capsulatum. J. Bacteriol, 89: 518-522.
22. Howard, D. H. 1973. Further studies on H^ capsulatum within macrophages from immunized animals. Infect. Immun. 577-588.
23. Howard, D. H. 1973. Fate of H. capsulatum in guinea pig PMNs. Infect. Immun. _8 : 412-418.
24. Howard, D. H. and Otto, V. 1976. Experiments on lymphocytes and mediated cellular immunity in murine histoplasmosis. Infect. Immun. % 6 : 226-231.
25. Hsu, H. S. and Kapral, F. A. 1960. The suppressed multiplication of tubercle baccilli within guinea pig macrophages. Amer. Rev. Resp. Dis. 9jL: 499-509.
26. Joklin, W., Willett, H., and Amos, D. 1980, Zinsser Microbiology 17th Ed. pp: 1327-1343.
27. Kashkin, K. P., Likholetov, S. M., and Lipnitsky, A. V. 1977. Studies on mediators of cellular immunity in experimental coccidiodomycosis, Sabouraudia, _1^: 59-68.
28. Kleinbaum, D., and Kupper, L. 1978. Applied Regression Analysis and Other Multivariate Methods. Duxbury Press, North Scituate, Mass. pp : 131-342.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. -45-
29. Laskin, A. I., and Lechavalier, H. 1972. Cellular Immunity in fungal infections, pp: 3-10. Macrophages and Cellular Immunity, CRC Press, Cleveland.
30. Laskin, A. I., and Lechavalier, H. 1972. Antimicrobial functions of Neutrophils, pp: 47-49. Macrophages and Cellular Immunity. CRC Press, Cleveland.
31. Lurie, H, 1963. Histopathology of Sporotrichosis. Arch. Pathol. 75: 421-437.
32. Lurie, M, B,, and Dannenberg, A. M., Jr. 1965. Macrophage functions on infectious diseases with inbred rabbits. Bact. Rev. 29: 466—476.
33. Lurie, H., and Still, J. 1969. The "capsule" of Sporotrichum schenckii and the evolution of the asteroid body, a light and electron microscopic study. Sabouraudia. 2.: 64-71.
34. Mackaness, G. B. 1962. Cellular resistance to infection. J. Exp. Med. 116: 381-388.
35. Mackaness, G. B., Blanden, R. V., and Collins, F. M. 1966. Host- parasite relations in mouse typhoid. J. Exp. Med. 124: 573-584.
36. Marcus. S., and Rambo, R. R. 1955. Comparisons of mice immunized against systemic mycosis. Proc. Soc. Amer. Bacteriol 92.
37. Mims, C. A., 1964. Aspects of the pathogenesis of virus disease. Bacterio. Rev. ^8: 36-56.
38. Hosteller, F., and Rourke, R. 1973. Sturdy Statistics, Nonpar- ametric and Order Statistics, pp: 224-229. Addison-Wesley, Reading, Mass.
39. Nelson, D. S., 1976. Immunobiology of the Macrophage. Academic Press. New York.
40. Newcomer, N. 1981. Antisporotrichic activity in mouse phagocytes. Master's Thesis, University of Montana.
41. Nie, Hull et. al. 1975. Statistical Package for Social Science: SPSS. 2nd Ed. McGraw-Hill, Chicago.
42. Pearsall, N., and Weiser, R, 1970. The Macrophage. Academic Press, New York.
43. Rippon, J. W. 1974. Introduction to medical mycology. Medical Mycology pp: 1-5. Saunders, Philadelphia.
44. Roitt, I. 1977. Essential Immunology. 3rd Ed. p 191. Blackwell Scientific Pub., Oxford, England.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. —46"
45. Schwartz, L. 1979. Compendium of Immunology pp 165-171. 2nd Ed. Von Norstrand, New York.
46. Snedecor, G., and Cochran, W. 1979. Statistical Methods. Iowa State Press. Âmes, Iowa. 7th Ed. pp: 298-391.
47. Thaler, M. S., Klausner, R. D., and Cohen, H. V. 1977. Medical Immunology, pp: 3-26. Lipincott, Philadelphia.
48. Youmans, G. P., and Youmans, A. S. 1969. Studies on acquired tuberculosis immunity. Cur. Top. Micro, and Immunol. 48: 129-177.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.